Sequence analysis of a 50 kb region between spoOH and rrnH on the Bacillus subtilis chromosome

Abstract

The 49630 bp sjpoOil-rmH region of the Bacillus subtilis genome has been fully sequenced. The sequence contains one partial and 62 complete ORFs, one partial and three complete rRNA genes and a cluster of six tRNA genes. The direction of the transcription and translation of 61 ORFs is the same as that of the movement of the replication fork. A homology search of 40 ORFs in newly determined sequence revealed that 27 of them had significant similarity to known proteins such as elongation factor G, elongation factor Tu, pseudouridine synthase I and ribosomal proteins. Two adjacent genes, ybaD and yh€, appeared to encode proteins belonging to the ATP-binding cassette (ABC) family.Keywords : Bacilhs stlbtiljs, genome sequencing, ribosomal protein gene cluster INTRODUCTIONWithin the framework of the international project for the sequencing of the entire Bacdlltls stlbtdh genome, we are responsible for sequencing a chromosomal region of about 150 kb between SpoOH(10") and pNEXT9 (24"). In this study, we determined the sequence of about 30 kb in the region between 10" and 15". We combined the results with known sequences and describe here a continuous 49630 bp sequence from spool3 (10') to rrtaH (15'). METHODSBacterial strains, phage and plasmids. B. subtilis 168 (trpC2), from our laboratory stock, was used as a template for PCR.Escherichia coli DH5a (stlpE44 lacU169 [480 lacZAM151 hsdR 17 recAl endAl gyrA96 tbi-I relAI) was used as the host for cloning with plasmid pTZ18R (Toyobo). The 1 phage K4F811 derived from EMBL 3a was supplied by F. Morohoshi (National Cancer Center, Tokyo, Japan).Sequence determination. All PCR products to be sequenced were self-ligated and fragmented by sonic oscillation, then 400-700 bp fragments were purified by means of agarose gel electrophoresis. These fragments were cloned into a vector (pTZ18R) and randomly overlapping libraries for shotgun sequencing were prepared. Inserts of pTZl8R were amplified by PCR using a forward (5' CGTCAAGGCGATTAAGTTG-GGTAA 3') and a reverse (5' TGCTTCCGGCTCGTATGT-TGTGTG 3') sequencing primer based on the vector sequence. The amplified PCR products were purified by precipitation with polyethylene glycol and used as templates for sequencing. Sequencing reactions were performed on both strands using the CATALYST Molecular Biology Labstation -Model 800 and Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems), and were then analysed with a Model 373A DNA Sequencer (Applied Biosystems).Data handling and computer analysis. DNA sequences were compiled using the GENETYX-MAC ATSQ (Software Development) and GENEWORKS programs (IntelliGenetics), We used sequence outputs only from the regions where every base was well separated (about 400 bp in one run). The compiled sequence was further searched for possible ORFs using GENETYX-MAC and GENEWORKS. A homology search of the identified ORFs was performed with the FASTA program using GENETYX-CD (Software Development) and a GenomeNet FASTA server in the Human Genome Center, Institute of Medical Science, University of Tokyo,...

Cloning, sequencing and transcriptional analysis of a Streptomyces coelicolor operon containing the rplM and rpsI genes encoding ribosomal proteins ScoL13 and ScoS9

“…The products of rplM and rpsI showed strong similarities with ribosomal proteins L13 and S9 from various prokaryotes and eukaryotes. The highest similarities (% amino acid sequence identity) of the rplM product were with L13 ribosomal proteins from Mycobacterium leprae (67.3%; GenBank accesion number U00020), Escherichia coli ( Yasumoto et al 1996). In accordance with the nomenclature established for ribosomal proteins (Wittman-Liebold et al 1990) we designated these two ribosomal proteins ScoL13 and ScoS9.…”

“…All the genes shown are transcribed in the same direction, from left to right. The sources of the sequences are as follows: S. coelicolor A3(2), this paper; Mycobacterium leprae, GenBank accesion number U00020; Bacillus subtilis, Yasumoto et al (1996); Mycoplasma pneumoniae, Himmelreich et al (1996);E. coli, Isono et al (1985) and GenBank accesion number U18997; Haemophilus somnus, Theisen and Potter (1992); Haloarcula marismortui, KroÈ mer and Arndt (1991) and Langer et al (1995) …”

Cloning and Characterization of the str Operon and Elongation Factor Tu Expression in Bacillus stearothermophilus

“…The region upstream of the ybxF gene is occupied by the 3Ј end of another ORF. Since it is 72% identical to the rpoC gene of B. subtilis that codes for the ␤Ј subunit of RNA polymerase (55), it was assigned to rpoC. The intergenic region between rpoC and ybxF is 136 nt long and contains a palindromic structure that most likely serves as a transcription termination signal for the rpoC gene (Fig.…”

“…Thus, E. coli and B. stearothermophilus represent two solutions of the same problem. According to DNA sequence data, it is likely that elements similar to those described here are also active in the B. subtilis (55) and B. halodurans str operon (50). Therefore, it is tempting to hypothesize that mechanisms of control of the EF-G/ EF-Tu ratio have common features among bacilli.…”

“…The following pairs of primers were used to amplify the respective regions of the str operon: for B. stearothermophilus, fus probe, PAN (5Ј-GCACAATGGAAAGGCCACCGC-3Ј) and VOK (5Ј-GC CCAGCCGGAGAAGACCGGG-3Ј), and ybxF probe (containing the entire ybxF gene), B. subtilis, fus probe, nucleotides (nt) 3772 to 3791 (forward primer) and nt 4019 to 4038 (reverse primer), and ybxF probe, nt 2429 to 2428 (forward primer) and nt 2661 to 2680 (reverse primer) (numbering is according to Yasumoto and coworkers [55]). …”